During normal development progenitor cells of many tissues undergo progressive restriction of pluripotency, epithelial-to- mesenchymal transition, proliferation, migration, and differentiation. Most, if not all, of these events involve modifications of cell-cell and cell-matrix adhesion, and abnormal modifications of these adhesion systems are often associated with the formation of tumors. The Eph family of receptor tyrosine kinases and their ligands, the ephrins, are frequently over-expressed in a wide variety of cancers, including breast, small-cell lung and gastrointestinal cancers, melanomas, and neuroblastomas. Using the Xenopus embryonic system, we have demonstrated that signaling mediated by the intracellular domain of ephrinB affects cell-cell adhesion, and that this activity can be modulated by interaction with an activated FGF receptor. The transmembrane ephrinB1 protein is a bi-directional signaling molecule that signals through its cytoplasmic domain to promote cellular movements into the eye field, whereas activation of the fibroblast growth factor receptor (FGFR) represses these movements and retinal fate. In Xenopus embryos, ephrinB1 plays a role in retinal progenitor cell movement into the eye field through an interaction with the scaffold protein Dishevelled (Dsh). We determined that FGFR-induced repression of retinal fate is dependent upon phosphorylation within the intracellular domain of ephrinB1, and disrupts the ephrinB1/Dsh interaction. This modulates retinal progenitor movement that is dependent on the planar cell polarity (PCP) pathway. These results provide mechanistic insight into how FGF signaling modulates ephrinB1 control of retinal progenitor movement within the eye field. Moreover, we found evidence that ephrinB1 signaling may regulate cell-cell junctions through a cell polarity complex in vivo. This study focused on assessing whether ephrinB1 is a mediator or modulator of cell-cell junction signaling in epithelial cells using the Xenopus system. We determined that the Par polarity complex protein, Par-6, which is a major scaffold protein required for establishing tight junctions, associates with ephrinB1 and is regulated by ephrinB1, resulting in the control of tight junctions. Using the epithelial cells of early stage Xenopus embryos, we showed that loss- or gain-of function of ephrinB1 can disrupt cell-cell contacts and tight junctions. This study reveals a mechanism where ephrinB1 competes with active Cdc42 for binding to Par-6, a scaffold protein central to the Par polarity complex (Par-3/Par-6/Cdc42/aPKC) and disrupts the localization of tight junction-associated proteins (ZO-1, Cingulin). This competition affects formation of tight junctions, and is regulated by tyrosine phosphorylation of ephrinB1. Finally, along with our colleagues in the Morrison laboratory, we have shown that ephrinB1 interacts with CNK1 in an EphB receptor- independent manner. In cultured cells, co-transfection of ephrinB1 with CNK1 increases JNK phosphorylation. EphrinB1/CNK1- mediated JNK activation is reduced by overexpression of dominant-negative RhoA, indicating that JNK activation is Rho-dependent. Overexpression of CNK1 alone is sufficient for activation of RhoA, however, both ephrinB1 and CNK1 are required for JNK phosphorylation. Co-IP analysis shows that ephrinB1 and CNK1 act as scaffold proteins that connect RhoA with JNK signaling components. Furthermore, adhesion to fibronectin and active Src overexpression increases ephrinB1/CNK1 binding, whereas inhibition of Src activity by a pharmacological inhibitor decreases not only ephrinB1/CNK1 binding, but also JNK activation. These data suggest that Src activity enhances ephrinB1/CNK1 interactions and downstream JNK activation. EphrinB1 overexpression increases cell motility of the HeLa cervical cancer cell line, however, CNK1 depletion by siRNA abrogates this cell migration and the accompanying JNK activation. Moreover, RhoA and JNK inhibitors suppress ephrinB1-mediated cell migration. Taken together, our findings suggest that CNK1 mediates ephrinB1-induced JNK activation and cell migration in cultured cancer cell lines.